Over 170 million persons are infected with Hepatitis C virus (HCV) and are at risk for liver failure and hepatocellular carcinoma. HCV is an enveloped RNA virus in the Flaviviridae family, which also includes the Flavivirus and Pestivirus genera. Molecular analysis of these latter genera has been facilitated by the ability to manipulate the complementary DNA genomes of these RNA viruses and to generate infectious virions that can be analyzed in cell culture. Viable chimeric Flaviviruses can be readily generated, and those expressing the envelope glycoproteins of pathogenic Flaviviruses within the nonpathogenic background of the 17D vaccine strain of the Yellow Fever Virus (YFV) have been shown to be safe and effective live vaccines. Efforts to understand the HCV life-cycle and to develop antiviral drugs and vaccines have been hindered by the inability to grow the virus in cell culture or in small animal models. Although restrictions on the in vitro propagation of HCV include those related to the interaction of the viral envelope glycoproteins with cellular receptor(s), additional barriers may include those that arise during the intracellular trafficking and assembly of the virion proteins. In this Small Research Grant proposal, we describe pilot studies to utilize the backbone of the strain 17D YFV vector to express the HCV envelope glycoproteins and develop a robust in vitro system to study HCV structure, function, and immunology. Specific Aims are: (1) to generate recombinant strain 17D YFV genomes bearing the HCV envelope glycoprotein genes, and (2) to assess the structural integrity of the HCV envelope glycoproteins and the ability of chimeric virion particles to be assembled and secreted. We will determine whether the chimeric virions are able to infect human hepatocellular carcinoma cells. Recombinant genomes may serve as a source of native HCV envelope glycoprotein complex for biochemical analysis. If chimeric virions are assembled and secreted, then the HCV envelope glycoproteins may mediate entry into appropriate target cells. Viable chimeric viruses will enable the study in cell culture of HCV binding and entry, as well as virion assembly and morphogenesis, and may provide a starting point towards the development of an attenuated HCV vaccine.